Translation-coupling systems

ABSTRACT

Disclosed are systems and methods for coupling translation of a target gene to a detectable response gene. A version of the invention includes a translation-coupling cassette. The translation-coupling cassette includes a target gene, a response gene, a response-gene translation control element, and a secondary structure-forming sequence that reversibly forms a secondary structure masking the response-gene translation control element. Masking of the response-gene translation control element inhibits translation of the response gene. Full translation of the target gene results in unfolding of the secondary structure and consequent translation of the response gene. Translation of the target gene is determined by detecting presence of the response-gene protein product. The invention further includes RNA transcripts of the translation-coupling cassettes, vectors comprising the translation-coupling cassettes, hosts comprising the translation-coupling cassettes, methods of using the translation-coupling cassettes, and gene products produced with the translation-coupling cassettes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) to U.S.Provisional Patent Application 61/289,739 filed Dec. 23, 2009, theentirety of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States government support awarded bythe following agencies: U.S. Department of Energy, DE-FC02-07ER64494.The United States government has certain rights in this invention.

FIELD OF THE INVENTION

The invention is directed to systems and methods for couplingtranslation of a target gene to a response gene in a host.

BACKGROUND

Production of recombinant proteins in hosts is a common process usedboth by researchers and commercial entities for the manufacture of alarge variety of proteins. In many instances, the protein is notproduced efficiently in particular host, so these proteins must beproduced through a different expression system. Unfortunately,conventional methods of determining the efficiency of protein expressionin a particular host require a number of time-consuming steps. Onemethod involves destroying the host, isolating proteins therefrom, andresolving the isolated proteins on a gel to detect the presence ofproduct bands at a particular molecular weight. Another method involvesdestroying the host, and performing a Western blot for the protein ofinterest. In addition to being time-consuming, both methods involveeither knowledge of the size of the protein of interest or an antibodythat specifically recognizes the expressed product. Further, theresearcher does not know about failed expression until all the steps areperformed. The ability to monitor protein translation directly in hosts,particularly in real-time, is limited.

One method of monitoring protein expression in real-time in hosts is tofuse the protein of interest directly to a fluorescent protein. Proteintranslation is measured through detection of fluorescence in the host.However, this method is limited in that fusion of the proteins canaffect activity of both the protein of interest and the fluorescentprotein and makes purification and isolation of the protein of interestdifficult.

Therefore, there remains a long-felt and unmet need for a system andmethod for quickly and reliably determining whether any given host iscapable of expressing the gene product of any given gene.

SUMMARY OF THE INVENTION

The present invention is directed to systems and methods for couplingtranslation of any desired target gene to a response gene in a host. Ifthe target gene is successfully translated, the response gene islikewise translated and, preferably, can be detected. If the target geneis not fully translated, the response gene is not translated, and thefunction of the protein encoded by the response gene cannot be detected.In an exemplary version of the invention, the coupling of translationbetween the target gene and the response gene occurs by including theresponse gene downstream of the target gene on the sametranslation-coupling cassette or vector. A response-gene translationcontrol element, such as a ribosome binding site, that controlstranslation of the response gene but not the target gene is includedbetween the target gene and the response gene in a sequence thatreversibly adopts a secondary structure when transcribed into RNA. Thesecondary structure occludes binding of ribosomes to the response-genetranslation control element or otherwise blocks its function such thattranslation of the response gene cannot occur. The sequence adopting thesecondary structure, however, is translationally linked to the targetgene such that full translation of the target gene unfolds the RNAsecondary structure in the RNA transcript, unmasks the response-genetranslation control element, and thereby permits translation of theresponse gene. This system can be used to screen for target-gene mutantsthat express in a particular host, determine culture conditions thatenable a host to efficiently express a target gene, or screen a DNAlibrary for factors that facilitate expression of a target gene.

Accordingly, one version of the invention includes atranslation-coupling cassette that comprises either a target gene or atarget-gene cloning site, a response gene or a response-gene cloningsite, a response-gene translation control element, and a secondarystructure-forming sequence that, when transcribed, reversibly forms asecondary structure that masks the response-gene translation controlelement, wherein at least part of the secondary structure-formingsequence is translationally linked with the target-gene cloning site orthe target gene.

The cloning sites can be multiple cloning sites, ligation-independentcloning sites, or any other cloning sites amenable to insertion of agene therein.

The response gene can be a screenable gene or a selectable gene.

The response-gene translation control element is preferably a ribosomebinding site. In some versions, the ribosome binding site comprises aShine-Dalgarno sequence or derivative thereof. In other versions, theribosome binding site comprises an AT-rich sequence. Thetranslation-coupling cassette may further include a linker disposedbetween the response-gene translation control element and the responsegene or the response-gene cloning site.

The translation-coupling cassette may further include a stop codonupstream, within, or downstream of the secondary structure-formingsequence, the stop codon being translationally linked with thetarget-gene cloning site or in-frame and translationally linked with thetarget gene. Including the stop codon within or downstream of thesecondary structure-forming sequence is preferred, and including thestop codon within the secondary structure-forming sequence is mostpreferred.

The translation-coupling cassette can further include a proteintag-encoding sequence, wherein the protein tag-encoding sequence istranslationally linked with the target-gene cloning site or in-frame andtranslationally linked with the target gene.

The secondary structure formed by the transcribed secondarystructure-forming sequence preferably includes a stem loop.

In a preferred version, the secondary structure-forming sequenceincludes at least a portion of a response-gene translation controlelement; a stop codon, wherein the stop codon is translationally linkedwith the target-gene cloning site or in-frame and translationally linkedwith the target gene; and at least a portion of a protein tag-encodingsequence, wherein the protein tag-encoding sequence is translationallylinked with the target-gene cloning site or in-frame and translationallylinked with the target gene. Exemplary secondary structure-formingsequences include bases 18-46 of SEQ ID NO:1, bases 26-60 of SEQ IDNO:2, bases 26-60 of SEQ ID NO:3, bases 26-60 of SEQ ID NO:4, and bases5-51 of SEQ ID NO:5.

In some versions of the invention, the translation-coupling cassette isincluded within a vector capable of being introduced in a host.

In some versions of the invention, the translation-coupling cassette isincluded within a host, preferably a prokaryotic host.

The invention also includes RNA molecules formed by transcription of anyof the versions of the translation-coupling cassette described herein.

The invention also includes methods of assessing expression of a targetgene using a translation-coupling cassette as described herein.

One method includes introducing a translation-coupling cassette into ahost and determining a level of expression of the response gene.

Another method further includes generating a mutated version of thetarget gene, cloning the mutated version of the target gene into atranslation-coupling cassette, introducing the translation-couplingcassette with the mutated version of the target gene into a host,determining the level of expression of the response gene in the host,and comparing the level of expression with that of non-mutated versions.

Another method includes culturing the host in a plurality of cultureconditions, determining a level of response-gene expression for each ofthe plurality of culture conditions, and comparing the levels ofresponse-gene expression for each of the plurality of cultureconditions.

Another method includes introducing into a host a translation-couplingcassette in a first vector and a clone from a DNA library, such as agenomic, cDNA, or metagenomic library in a second vector, to screen forfactors that enhance target-gene expression.

Among other advantages, the methods of assessing protein translationdescribed herein are quick and involve few processing tasks. The methodsdo not impact the activity of the protein of interest or complicatesubsequent isolation and purification methods.

The invention also includes gene products fabricated by any of themethods or procedures described herein. One version includes a geneproduct fabricated by translating a gene from a transcript of atranslation-coupling cassette as described herein, wherein the gene isselected from the group consisting of a target gene and a response gene.

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention made in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts an mRNA transcript of a translation-coupling cassette ofthe present invention with full translation of a target gene, disruptionof RNA secondary structure, and consequent translation of a responsegene.

FIG. 1B depicts the mRNA transcript of FIG. 1A with incompletetranslation of the target gene, masking of a ribosome binding site byRNA secondary structure, and inhibition of response gene translation.

FIG. 2A depicts a portion (bases 10-51) of a translation-couplingcassette identified herein as “TC1” (SEQ ID NO: 1; see Table 1), whichincludes a downstream portion of a cloning site (BglII; bases 10-15), aprotein tag-encoding sequence (6x-His tag; bases 16-33), a stop codon(bases 34-36), a response-gene translation control element (RBS; bases35-40), a linker (bases 41-48), a start codon of a response gene (Cm^(R)start; bases 49-51), and a secondary structure-encoding sequence (bases18-46).

FIG. 2B depicts a portion (bases 19-64) of a translation-couplingcassette identified herein as “TC2” (SEQ ID NO: 2; see Table 1), whichincludes a downstream portion of a cloning site (EcoRI; bases 19-24), aprotein tag-encoding sequence (6x-His tag; bases 25-42), a stop codon(bases 43-45), a response-gene translation control element (RBS; bases48-53), a linker (bases 54-61), a start codon of a response gene (Cm^(R)start; bases 62-64), and a secondary structure-encoding sequence (bases26-60).

FIG. 2C depicts a portion (bases 19-64) of a translation-couplingcassette identified herein as “TC3” (SEQ ID NO: 3; see Table 1), whichincludes a downstream portion of a cloning site (SpeI; bases 19-24), aprotein tag-encoding sequence (6x-His tag; bases 25-42), a stop codon(bases 43-45), a response-gene translation control element (RBS; bases48-53), a linker (bases 54-61), a start codon of a response gene (Cm^(R)start; bases 62-64), and a secondary structure-encoding sequence (bases26-60).

FIG. 2D depicts a portion (bases 19-64) of a translation-couplingcassette identified herein as “TC4” (SEQ ID NO: 4; see Table 1), whichincludes a downstream portion of a cloning site (SpeI; bases 19-24), aprotein tag-encoding sequence (6x-His tag; bases 25-42), a stop codon(bases 43-45), a response-gene translation control element (RBS; bases48-53), a linker (bases 54-61), a start codon of a response gene(Kan^(R) start; bases 62-64), and a secondary structure-encodingsequence (bases 26-60).

FIG. 2E depicts a portion (bases 1-53) of another translation-couplingcassette identified herein as “TC5” (SEQ ID NO: 5; see Table 1), whichincludes a downstream portion of a cloning site (SpeI; bases 4-9), aprotein tag-encoding sequence (6x-His tag; bases 10-27), a stop codon(bases 28-30), a response-gene translation control element (RBS; bases29-34), a linker (bases 35-46), an upstream coding sequence of aresponse gene (kan^(R) start and onward; bases 47-53), and a secondarystructure-encoding sequence (bases 5-51).

FIGS. 3A, 3B, and 3C show growth curves, fluorescence curves, and anSDS-PAGE analysis of purified target-gene protein product, respectively,from E. coli cells transformed with the pTC1-RFP plasmid (see Tables 1and 2) and grown in the presence of the shown concentrations (μg/ml) ofchloramphenicol.

FIGS. 4A and 4B show growth and fluorescence curves, respectively, fromE. coli cells transformed with the pTC1-RFP* plasmid (see Tables 1 and2) and grown in the presence of the shown concentrations (μg/ml) ofchloramphenicol.

FIGS. 5A and 5B show growth and fluorescence curves, respectively, fromE. coli cells transformed with the pTC2-RFP plasmid (see Tables 1 and 2)and grown in the presence of the shown concentrations (μg/ml) ofchloramphenicol.

FIGS. 6A and 6B show growth and fluorescence curves, respectively, fromE. coli cells transformed with the pTC2-RFP* plasmid (see Tables 1 and2) and grown in the presence of the shown concentrations (μg/ml) ofchloramphenicol.

FIGS. 7A and 7B show growth and fluorescence curves, respectively, fromE. coli cells transformed with the pTC4-RFP plasmid (see Tables 1 and 2)and grown in the presence of the shown concentrations (μg/ml) ofkanamycin.

FIGS. 8A and 8B show growth and fluorescence curves, respectively, fromE. coli cells transformed with the pTC4-RFP* plasmid (see Tables 1 and2) and grown in the presence of the shown concentrations (μg/ml) ofkanamycin.

FIG. 9 depicts the predicted domain structure of a previouslyuncharacterized and unexpressed gene (“Gene X”). Eight predicteddomains, Domains 1-8, are shown.

FIG. 10 shows a growth curve from E. coli cells transformed with thepTC4-D1-D8 plasmid (see Tables 1 and 2), comprising full-length Gene Xand grown in the presence of the shown concentrations (μg/ml) ofkanamycin.

FIGS. 11A and 11B a show a bacterial growth curve and an SDS-PAGEanalysis of purified target-gene protein product, respectively, from E.coli cells transformed with the pTC4-D1-D2 plasmid (see Tables 1 and 2),comprising only the portion of Gene X encoding Domains 1 and 2, andgrown in the presence of the shown concentrations (μg/ml) of kanamycin.

FIGS. 12A and 12B a show a bacterial growth curve and an SDS-PAGEanalysis of purified target-gene protein product, respectively, from E.coli cells transformed with the pTC4-D3-D5 plasmid (see Tables 1 and 2),comprising only the portion of Gene X encoding Domains 3-5, and grown inthe presence of the shown concentrations (μg/ml) of kanamycin.

FIGS. 13A and 13B a show a bacterial growth curve and an SDS-PAGEanalysis of purified target-gene protein product, respectively, from E.coli cells transformed with the pTC4-D6-D8 plasmid (see Tables 1 and 2),comprising only the portion of Gene X encoding Domains 6-8, and grown inthe presence of the shown concentrations (μg/ml) of kanamycin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises a translation-coupling cassette thatcouples full translation of a target gene (and translationally linkedsequences, excluding stop codons) to the expression of a selectable,screenable, and/or otherwise detectable response gene. Versions of thetranslation-coupling cassette accordingly include either a target geneor a target-gene cloning site for insertion of a target gene, togetherwith a response gene or a response-gene cloning site for insertion of aresponse gene.

Whether used with reference to a target gene or a response gene, “gene”refers to a nucleic acid sequence that includes at least one start codonfollowed by a coding sequence for at least one polypeptide. For thepurposes herein, “gene” may or may not include a stop codon, a promoter,enhancers, or other elements required for its expression (see below). Agene may include introns in addition to exons, particularly if derivedfrom eukaryotic genomic DNA. Genes that include introns are preferablyexpressed in eukaryotic hosts or other expression systems capable ofexcising the introns. Genes configured for being expressed inprokaryotic hosts preferably do not include introns. As used herein,references to a “gene,” such as a “target gene” or a “response gene,”refers to sequences on a DNA translation-coupling cassette and anycorresponding sequences on a transcript thereof, unless explicitlystated otherwise or otherwise implied by the context.

“Target gene” refers to any gene encoding a polypeptide that is desiredto be expressed for any purpose. The target gene is typicallyheterologous to a host but may also be native to the host. If the targetgene is native to the host, its expression may be desired underconditions different than those under which it is normally expressed, orits expression may be desired at levels greater than or less than thoseat which it is normally expressed. The target gene can be obtained byany method, including cloning from genomic or cDNA libraries, subcloningfrom gene fragments, or, if the nucleotide sequence is known, directsynthesis. Techniques for cloning and subcloning DNA and generatinggenomic DNA and cDNA libraries are widely known to those of skill in theart. DNA synthesis can be performed by any of several commerciallyavailable synthesizers known to those skilled in the art. In a preferredversion of the invention, the target gene does not include its ownpromoter and relies on a promoter elsewhere in a vector harboring thetranslation-coupling cassette to initiate its transcription. It is alsopreferred that the target gene does not include its own stop codon andinstead relies on a stop codon elsewhere in the translation-couplingcassette, such as in a secondary structure-forming sequence (see below),to stop its translation. To facilitate read-through of the ribosome to astop codon in the translation-coupling cassette, the target genepreferably does not include its own 3′ untranslated region (3′ UTR). Asused herein, “translation of the target gene” refers to translation ofthe coding sequence of the target gene on an RNA transcript of thetranslation-coupling cassette.

In place of a target gene, the translation-coupling cassette may includea target-gene cloning site configured for insertion of a target gene.The cloning site may comprise any sequence of nucleic acid residuesamenable to insertion, by any method (i.e., ligation,ligation-independent cloning, homologous recombination, ligation, etc.),of a target gene therein. In one version, the cloning site includes oneor more restriction sites for digesting with an appropriate restrictionenzyme and inserting a desired target gene therein. For example, thecloning site may comprise a multiple cloning site (MCS), also called apolylinker. An MCS is a short segment of DNA containing several (up to20 or more) different restriction sites. Examples of restriction sitesthat can comprise an MCS include, without limitation, EcoRI, BglII,ClaI, PvuI, BamHI, KpnI, XbaI, SalI, HincII, PstI, and SpeI. MCSs are astandard feature of many commercially available plasmids. See, forexample, Clark D P (2005), “Molecular Biology,” Academic Press, ISBN0121755517, at page 611. Any of these MCSs are suitable for use in thepresent invention. It is preferable that any restriction site in thecloning site occurs only once within a given translation-couplingcassette or plasmid containing it.

The cloning site may also comprise a ligation-independent cloning site.Design of ligation-independent cloning sites and methods ofligation-independent cloning are well known in the art. See, e.g.,Cabrita et al. BMC Biotechnology (2006) 6:12. Briefly, the cloning siteis designed around restriction sites such that the 16-18 or so basessurrounding the restriction site are missing one of the fournucleotides, such as adenine (A). One example of ligation-independentcloning with such a site is T4 polymerase-mediated ligation-independentcloning (T4P-LIC). The vector comprising the translation-couplingcassette is first linearized by a restriction enzyme or enzymes at thecloning site. The vector is treated with T4 DNA polymerase in thepresence of only a single type of nucleotide, such as dATP, for example.The exonuclease activity degrades the exposed 3′ ends of the linearizedvector until reaching the first A base, at which point the T4 DNApolymerase's exonuclease and polymerase activities balance each otherout, leaving the vector with 16-18-base, 5′ overhangs. The insert isamplified by PCR using oligos with tails that, when treated with T4polymerase and dTTP, create overhangs compatible with those on thevector. The vector and insert are then annealed together andtransformed. The long sticky ends on the plasmid and insert aresufficient to hold the plasmid and insert together, allowing them to betransformed without prior ligation. The existing nicks are then repairedby ligases in the host cell. If the left and right sticky ends on theplasmid are different, the cloning is directional.

Other cloning sites include BioBrick® cloning sites (Shetty et al. J.Biol Eng. (2008) Apr. 14; 2:5).

Non-limiting examples of cloning sites for use in the present inventionand the placement of the target-gene cloning site with respect to theother elements in the translation-coupling cassette include bases 1-15of SEQ ID NO:1, bases 1-24 of SEQ ID NO:2 (ligation-independent cloningcompetent), bases 1-24 of SEQ ID NO:3, bases 1-24 of SEQ ID NO:4, andbases 1-9 of SEQ ID NO:5. FIGS. 2A-2E show downstream restriction sitesof these cloning sites.

Regardless of the type of cloning site used, steps can and should betaken to ensure that any target gene inserted in the cloning site isin-frame with any sub-elements that are translationally linked with thecloning site, such as a protein tag-encoding sequence and a stop codon(see below). This can be done with appropriate cloning strategies, whichare well-known in the art.

The target gene or target-gene cloning site is preferably operablylinked to a sequence encoding an element responsible for controllingtranslation of the existing target gene or any subsequently insertedtarget gene, but not the response gene. As used herein, “controllingtranslation” refers to any mechanism of inducing translation, includingbut not limited to promoting initiation of translation. Such an elementis preferably a ribosome binding site, examples of which are discussedin further detail below.

“Response gene” also refers to any gene encoding a polypeptide that isdesired to be expressed for any purpose. In a preferred version of theinvention, the response gene is any gene that encodes a product capableof being detected upon expression. It is preferable that such detectionis amenable to high-throughput detection, real-time detection, orautomated detection. Successful expression of a response gene confersthe ability to distinguish between hosts (and their progeny) thatexpress the response gene and hosts (and their progeny) that do notexpress the response gene. Examples of genes that are capable of beingdetected include selectable response genes and screenable responsegenes. As used herein, “translation of the response gene” refers totranslation of the coding sequence of the response gene on an RNAtranscript of the translation-coupling cassette.

Selectable response genes are genes that confer the ability to select ahost expressing the gene from those that do not express the gene.Selectable response genes typically confer resistance to a selectionagent that kills hosts not expressing the gene. Non-limiting examples ofselectable response genes include antibiotic resistance genes;auxotrophic complementation genes, herbicide tolerance genes; metaltolerance genes; and drug resistance genes, such as that providingresistance to methotrexate (see e.g., U.S. Pat. No. 5,179,017), amongothers. Suitable antibiotic resistance genes include any antibioticresistance marker now known or developed in the future, including(without limitation) markers that confer resistance to ampicillin (e.g.,beta-lactamase [bla, TEM-1]), hygromycin (e.g., hygromycinphosphotransferase [aphIV, hpt]), kanamycin, neomycin (e.g., neomycinphosphotransferase II [nptII, APH(3′)-II]), chloramphenicol (e.g.,chloramphenicol acetyltransferase [cm^(R), cat]), tetracycline(tet^(R)), and the like. Suitable auxotrophic complementation genesinclude those involved in DNA-precursor, amino-acid, or cell-wallbiosynthetic pathways. Examples of suitable auxotrophic complementationgenes include, without limitation, asd (encoding aspartatebeta-semialdehyde dehydrogenase), thyA (encoding thymidylatesynthetase), glnA (encoding glutamine synthase), leuD (encodingisopropylmalate isomerase small subunit), pyrF (encodingorotidine-5′-phosphate decarboxylase), proC (encodingpyrroline-5-carboxylate reductase), glyA (encoding serine hydroxymethyltransferase), and nadC (encoding quinolinic acidphosphoribosyltransferase), These genes can complement hosts that areauxotrophic for particular nutrients or factors as a result of adisruption of a corresponding chromosomal gene in the host.

Screenable response genes are genes that confer the ability to identifya host expressing the gene and distinguish such hosts from those that donot express the gene. Screenable response genes typically produceproducts that emit a signal, such as a light signal, in response to astimulus or a chemical precursor. For example, some response-geneproducts emit light signals upon irradiation with certain wavelengths oflight. Other response-gene products catalyze a reaction wherein thereaction product emits light. The signals produced by the screenableresponse-gene products are preferably unique signals and are preferablydetectable in real-time. These genes include (by way of example only)genes that express chromophoric proteins; genes that signaling proteinsand/or regulatory proteins; genes that express detectable surfacemarkers such as CD8; genes that express fluorescent proteins such asgreen-fluorescent protein, red-fluorescent protein, etc.; luciferasegenes; the lacZ gene; the alkaline phosphatase gene; and variantsthereof.

Other various examples of response genes include genes involvedmutagenesis, carcinogenesis, or onset or alleviation of any givendisease state; genes encoding enzymes, such as the CAD gene (see, e.g.,Wahl et al., Somat. Cell Mol. Genet. 12:339 (1986), among others; genesinvolved in metabolism, such as amino acid metabolism; genes influencingphytohormone production, and the like.

Non-limiting examples of response genes and their placement with respectto the other elements within the translation-coupling cassette includethe chloramphenicol coding sequences at positions 49-708 in SEQ ID NO:1(see FIG. 2A), positions 62-721 in SEQ ID NO:2 (see FIG. 2B), positions62-721 in SEQ ID NO:3 (see FIG. 2C), and the kanamycin coding sequenceat positions 62-871 in SEQ ID NO:4 (see FIG. 2D). See also the startcodon and early coding sequence for the kanamycin resistance gene atpositions 47-49 in SEQ ID NO:5 (see FIG. 2E).

In some versions of the invention, the translation-coupling cassetteincludes a response-gene cloning site in place of a response gene forinclusion of any response gene desired by a user. The response-genecloning site may comprise any sequence of nucleic acid residues amenableto insertion, by any method, of a response gene therein and may includeall those described herein for the target-gene cloning site. As with thetarget-gene cloning site, the response-gene cloning site may comprise asingle restriction site, an MCS, or a ligation-independent cloning site.If the translation-coupling cassette comprises both a target-genecloning site and a response-gene cloning site, it is preferred that thetarget-gene cloning site and the response-gene cloning site aredifferent and complementary.

In a preferred version of the invention, the inserted target gene, theinserted response gene, and any intervening sequences therebetween areoperably connected to only one promoter, wherein the one promoter iscapable of inducing transcription of the inserted target gene, theinserted response gene, and the intervening sequences. In this manner,the translation-coupling cassette is capable of producing apolycistronic RNA transcript and constitutes an artificial operon. Thepromoter is preferably disposed upstream of the target gene ortarget-gene cloning site. The promoter can be included in a vector (seebelow) harboring the translation-coupling cassette at a positionupstream of the target gene or the target-gene cloning site forsubsequent operable linkage with an inserted target gene. Alternatively,the promoter can be incorporated into the target-gene cloning site ofthe translation-coupling cassette along with the target gene, providedthat a vector harboring the translation-coupling cassette does notalready have a promoter operably connected to the target-gene cloningsite. Any promoter suitable for transcription in a host in which thetranslation-coupling cassette is intended to be introduced may be used(see below). Depending on the host, any of a number of suitable controlelements, such as transcription enhancer elements (see e.g., Bitter etal. (1987) Methods in Enzymology, 153:516- 544), can be included in thetranslation-coupling cassette if not already included in a vectorharboring the translation-coupling cassette. Alternatively, the controlelements can be in the vector harboring the translation-couplingcassette.

The response gene is preferably operably linked to a sequence encoding aresponse-gene translation control element. As used herein,“response-gene translation control element” refers to a sequenceresponsible for controlling translation of the response gene.“Response-gene translation control element” is generally used herein torefer both to the element on the DNA translation-coupling cassette andthe corresponding element on the on the RNA transcript thereof, unlessexplicitly stated otherwise or otherwise implied by the context. Theresponse-gene translation control element is distinct from the elementcontrolling translation of the target gene. The response-genetranslation control element can comprise any sequence known or hereafterdiscovered that induces translation of a gene when exposed but does notinduce translation of a gene when masked or otherwise unexposed.

In a preferred version of the invention, the response-gene translationcontrol element is a ribosome binding site. A ribosome binding site is asequence on an RNA transcript that binds a ribosome in initiatingpolypeptide translation. Examples of ribosome binding sites include theShine-Dalgarno sequence and an AU-rich sequence.

The Shine-Dalgarno sequence has the consensus sequence AGGAGG and isgenerally located about 8-10 bases upstream of the start codon of theresponse gene whose translation it controls. Many variants of the AGGAGGconsensus are known in the art and can be used as a ribosome bindingsite in the translation-coupling cassette described herein. For example,the sequence in E. coli is AGGAGGU. Other variants include GGAG,GGGUGGU, AGGA, UAAGGA, GGAGG, GGGGU, AGGAG, GAG, AGGG, and TGGTGG. Seealso, Shultzaberger et al. Journal of Molecular Biology (2001)313(1):215-228. The region between the downstream end of theresponse-gene translation control element, such as the ribosome bindingsite, and either the response-gene cloning site or the start codon ofthe response gene is referred to herein as a “linker.” A linker for theShine-Dalgarno sequence or variants thereof can include a number ofbases from about 1 to about 20, inclusive, such as about 8-10 bases, andcan comprise any sequence. The length of the linker can be optimized incertain hosts for optimal translation of the response gene.

Non-limiting examples of response-gene translation control elements asribosome binding sites and their placement with respect to the otherelements in the translation-coupling cassettes of the present inventionare included herein as bases 35-40 in SEQ ID NO:1 (AGGAGG; see FIG. 2A),bases 48-53 in SEQ ID NO:2 (TGGTGG; see FIG. 2B), bases 48-53 in SEQ IDNO:3 (TGGTGG; see FIG. 2C), bases 48-53 in SEQ ID NO:4 (TGGTGG; see FIG.2D), and bases 29-34 in SEQ ID NO:5 (AGGAUG; see FIG. 2E).

Non-limiting examples of linkers, their lengths, and their placementwith respect to the other elements in the translation-coupling cassettesof the present invention are included herein as bases 41-48 in SEQ IDNO:1 (see FIG. 2A), bases 54-61 in SEQ ID NO:2 (see FIG. 2B), bases54-61 in SEQ ID NO:3 (see FIG. 2C), 54-61 in SEQ ID NO:4 (see FIG. 2D),and bases 35-46 in SEQ ID NO:5 (see FIG. 2E).

AU-rich sequences serve as binding sites for the 51 ribosomal protein ofthe prokaryotic ribosome. Examples of various AU-rich sequences includeUAUAAAA, AACACUA, UAUAAAA, AACACUA, AAACACAU, AAUAAAAU, UUAACCUUA, andUUAACUUUA. Other AU-rich sequences are well known in the art. See, e.g.,Durand et al. Nucleic Acids Research (2006) 34(22):6549-6560 and Indiaet al. J. Bacteriol. (2007) 189(11):4028-4037. Any of these sequencescan be used in the present invention. The AU-rich sequence is preferablyseparated from the start codon of the response gene by a linkercomprising a number of bases from about 1 to about 50 bases, inclusive,such as about 15 to about 30 bases.

The translation-coupling cassette of the present invention preferablyincludes a secondary structure-forming sequence. This is a sequence onthe translation-coupling cassette that, when transcribed into acorresponding RNA transcript, reversibly forms a secondary structure.The secondary structure-forming sequence is preferably disposed on thetranslation-coupling cassette with respect to the response gene-controlelement such that formation of the secondary structure masks theresponse gene-control element, thereby inhibiting translation of theresponse gene. If the response gene-control element is a ribosomebinding site, formation of the secondary structure blocks ribosomebinding thereto and thereby prevents initiation of translation. In apreferred version of the invention, at least part of the responsegene-control element overlaps with the secondary structure-formingsequence. However, other configurations are acceptable. For example, theresponse gene-control element may be wholly subsumed within thesecondary structure-forming sequence, or the secondary structure-formingsequence may be wholly subsumed within the response gene-controlelement. In yet another configuration, the response gene-control elementand the secondary structure-forming sequence do not overlap at all, solong as the elements are mutually disposed such that the presence of thesecondary structure on the translation-coupling cassette transcriptoccludes ribosome binding to the ribosome binding site or otherwiseinhibits induction of translation via the response gene-control element.

The secondary structure-forming sequence may comprise any sequence ofnucleotides that yields a secondary structure that masks, occludes, orotherwise inhibits the function of the response gene-control element ininducing expression. Examples of various elements of RNA secondarystructure include stacks, hairpin loops, bulges, internal loops,junctions, and multiloops. Secondary structure-forming sequences can begenerated using RNA structure prediction software. Many versions of suchsoftware are well-known in the art and include, by way of example,RNAfold, RNAshapes, Pknots, Mfold, among others. In a preferred versionof the invention, the formed secondary structure comprises a stem loop,also known as a hairpin loop or hairpin.

Formation of a stem loop can be accomplished when the DNA sequencedownstream of the target gene is designed to cause formation of anintra-molecular double-helix in the corresponding RNA transcript. Thisoccurs when two regions of the same strand base-pair to form a doublehelix that ends in an unpaired loop. The double helix forms the “stem”portion of the stem loop and the unpaired loop forms the loop portion.Sequences that yield stem-loop structures typically include a downstreamsegment that is substantially a reverse complement of an upstreamsegment, which together enable intra-molecular base pairing. Forexample, an AAGC upstream sequence may base pair with a GCUU downstreamsequence. The stability of this helix is determined by its length, thenumber of mismatches or bulges it contains (a small number aretolerable, especially in a long helix), and the base composition of thepaired region. Pairings between G and C have three hydrogen bonds andare more stable compared to A-U pairings, which have only two hydrogenbonds. The stability of the unpaired loop portion of the stem-loopstructure also influences the formation of the stem-loop structure.Loops that are fewer than three bases long are sterically hindered anddo not form. Large loops with no secondary structure of their own (suchas pseudo-knot pairing) are also unstable. Optimal loop length tends tobe from about four (4) to about ten (10) bases. Base stackinginteractions, which align the pi orbitals of the bases' aromatic ringsin a favorable orientation, also promote stability. One common loophaving the sequence UUCG is particularly stable due to the base-stackinginteractions of its component nucleotides.

Preferred secondary structure-forming sequences includereverse-complementary sequences separated by from about four to aboutten bases that define a loop portion when the reverse-complementarysequences bind to define the stem portion of the stem-loop structure.Non-limiting examples of secondary structure-forming sequences,specifically stem loops, and their positions with respect to the otherelements in the translation-coupling cassette are included as positions18-46 in SEQ ID NO:1 (see FIG. 2A), positions 26-60 in SEQ ID NO:2 (seeFIG. 2B), positions 26-60 in SEQ ID NO:3 (see FIG. 2C), positions 26-60in SEQ ID NO:4 (see FIG. 2D), and positions 5-51 in SEQ ID NO:5 (seeFIG. 2E).

In the preferred version of the invention, at least a portion of thesecondary structure-forming sequence is translationally linked with thetarget-gene cloning site or the target gene. One version of atranslation-coupling cassette having at least a portion of the secondarystructure-forming sequence translationally linked with the target-genecloning site or target gene includes a stop codon within or downstreamof the secondary structure-forming sequence. Translation of apre-existing target gene or target gene subsequently inserted in thetarget-gene cloning site will comprise translation of the target gene,any intervening sequences between the target gene and the secondarystructure-forming sequence, and portions or all of the secondarystructure-forming sequence up to the stop codon. The stop codon and anypre-existing target gene in the expression vector should be in-frame.Similarly, any target gene inserted in a target-gene cloning site shouldbe inserted in-frame with the stop codon.

In a preferred version of the invention, the stop codon is includedwithin the secondary structure-forming sequence. Further, if thesecondary structure-forming sequence is configured to form a stem loop,the stop codon may be included within an upstream segment of the stemportion of the stem loop, within the loop portion of the stem loop, orwithin a downstream segment of the stem portion of the stem loop. (SeeFIG. 1B for loop portion 34 of the stem loop 30, upstream segment 32,and downstream segment 36.) This positioning ensures unwinding of thestem loop and unmasking of the response-gene translation control elementduring translation of the target gene (see examples). The preferredlocations of the stop codon in the stem loop and its positioning withrespect to the response-gene translation control element are as shown inFIGS. 2A-E.

In yet other versions, the stop codon may be included upstream of thesecondary structure-forming sequence.

Non-limiting examples of stop codons translationally linked with thetarget genes or target gene cloning sites and their positions withrespect to the other elements in the translation-coupling cassette areincluded as bases 34-36 in SEQ ID NO:1 (see FIG. 2A), bases 43-45 in SEQID NO:2 (see FIG. 2B), bases 43-45 in SEQ ID NO:3 (see FIG. 2C), bases43-45 in SEQ ID NO:4 (see FIG. 2D), and bases 28-30 in SEQ ID NO:5 (seeFIG. 2E).

The translation-coupling cassette may further include a sequenceencoding a protein tag that is either translationally linked with atarget-gene cloning site or in-frame and translationally linked with atarget gene. Thus, translation of the pre-existing target gene or anytarget gene inserted in-frame in the target-gene cloning site includestranslation of the protein tag as a sub-domain of the translatedtarget-gene product. Fusion of a protein tag in this manner can beuseful in any number of applications using the product expressed fromthe target gene, including purification via immunoprecipitation oraffinity chromatography, detection via immunoblotting, or real-timedetection. Non-limiting examples of suitable protein tags include chitinbinding protein (CBP), maltose binding protein (MBP),glutathione-S-transferase (GST), poly(His) tag, thioredoxin (TRX),poly(NANP), FLAG tag, V5 tag, c-myc tag, HA tag, fluorescent tags suchas green fluorescent protein (GFP) or red fluorescent protein (RFP),biotin ligase tag, isopeptag, biotin carboxyl carrier protein, S tag,strep tag, and SBP tag.

In a preferred version of the invention, at least a portion of theprotein tag-encoding sequence is included as part of the secondarystructure-forming sequence. A preferred sequence for this purpose is asequence encoding a poly(His) tag, such as a 6x-His tag.

Non-limiting examples of protein-tag encoding sequences (encoding 6x-Histags) translationally linked with the target genes or target genecloning sites and their positions with respect to the other elements inthe translation-coupling cassette are included as bases 16-33 in SEQ IDNO:1 (see FIG. 2A), bases 25-42 in SEQ ID NO:2 (see FIG. 2B), bases25-42 in SEQ ID NO:3 (see FIG. 2C), bases 25-42 in SEQ ID NO:4 (see FIG.2D), and bases 10-27 in SEQ ID NO:5 (see FIG. 2E).

Operation of an exemplary version of the translation-coupling cassetteof the present invention is as follows. The exemplarytranslation-coupling cassette includes a target gene, a response gene, aresponse-gene translation control element, and a secondarystructure-forming sequence, wherein at least part of the secondarystructure-forming sequence is translationally linked with the targetgene. Transcription of such a translation-coupling cassette generates anmRNA transcript with corresponding genetic elements, wherein thesecondary structure in the secondary structure-forming sequence isformed in the absence of active or complete translation of the targetgene. When the target gene is fully translated, the ribosome alsotranslates any downstream sequences translationally linked to the targetgene up to the first in-frame stop codon. In the process of translation,the ribosome also translocates along the same sequences, unwinding theRNA secondary structure that reversibly forms in the absence oftranslation. Flattening the RNA secondary structure exposes theresponse-gene translation control element and permits translation of theresponse gene. In cases in which the response-gene translation controlelement is a ribosome binding site, flattening of the RNA structurepermits access of the ribosome to bind to the ribosome binding site toinitiate translation. In this manner, full translation of the targetgene results in translation of the response gene. Disrupted or otherwiseincomplete translation of the target gene, however, results in inhibitedtranslation of the response gene due to masking of the response-genetranslation control element.

Hosts that fully translate the target gene are identified by detectingthe presence of the response-gene protein product, either directly orindirectly. For example, if the response gene is an antibioticresistance response gene, the transformed hosts that fully translate thetarget gene are resistant to the corresponding antibiotic. If theresponse gene is a fluorescent protein response gene, the transformedhosts that fully translate the target gene fluoresce at the wavelengthcorresponding to the response-gene product. If the response gene is anenzyme-encoding gene, the transformed hosts that fully translate thetarget gene demonstrate the enzymatic activity of the response-geneproduct. If the response gene is a gene encoding a regulatory protein,the transformed hosts that fully translate the target gene demonstratethe additional regulatory activity.

If the product of the target gene is not fully translated, the responsegene is also not translated because the secondary structure in the mRNAtranscript remains intact. The secondary structure in the mRNA thereforemasks or occludes the response-gene translation control element.Transformed hosts that do not successfully translate the target gene donot display the characteristics of the gene product of the responsegene. That is, if the response gene is an antibiotic resistance gene,the transformed hosts are sensitive to antibiotic rather than resistantto it. If the response gene is a fluorescent protein response gene, thetransformed hosts do not fluoresce at the wavelength corresponding tothe response-gene product. If the response gene is an enzyme-encodinggene, the transformed cells do not demonstrate the enzymatic activity ofthe response-gene product. If the response gene encodes a regulatoryprotein, the transformed hosts do not demonstrate the additionalregulatory activity.

An illustrative mRNA transcript 10 of a translation-coupling cassette ofthe present invention is depicted in FIGS. 1A and 1B. The depictedtranscript 10 includes a target gene 12 (AUG . . . Gene X) followed by adownstream portion of a target-gene cloning site 17 and atranslationally linked and in-frame protein tag-encoding sequence 14(6x-His) and stop codon 16 (Stop). A separate ribosome binding sitecontrolling translation of the target gene 12 is not shown. The depictedtranscript 10 also includes a response gene 20 (AUG . . . Cm^(R) . . .TGA) preceded by a ribosome binding site 18 (RBS) controlling expressionof the response gene 20. Interspersed between the ribosome binding site18 and the response gene 20 is a linker 19. The protein tag-encodingsequence 14, the stop codon 16, and the ribosome binding site 18together form a secondary structure-forming sequence 28.

FIG. 1A depicts complete translation of the target gene 12 coupled withtranslation of the response gene 20. Shown are ribosomes 22 translatingthe target gene 12 through to the translationally linked proteintag-encoding sequence 14 and translocating to the stop codon 16. Thecomplete translation of the target gene 12 disrupts RNA secondarystructure, such as a stem loop 30 that otherwise occludes the ribosomebinding site 18 of the response gene 20 (see FIG. 1B). Disrupting thesecondary structure exposes the ribosome binding site 18 and therebyenables translation of the response gene 20. As a result, both thetarget-gene product 24 and the response-gene product 26 are fullytranslated. Successfully transformed hosts are then selected and/oridentified based on the characteristics of the expressed gene product 26of the response gene 20.

FIG. 1B depicts interrupted or incomplete translation of the target gene12. Here, the target gene 12 is not fully translated to itscorresponding gene product 24, and the ribosome 22 fails to translocatethrough to the protein tag-encoding sequence 14 and the stop codon 16.The failure to fully translocate enables formation of a secondarystructure. The depicted secondary structure is a stem loop 30 thatincludes a stem portion, defined by an upstream segment 32 and adownstream segment 36, and a loop portion 34 between the upstreamsegment 32 and the downstream segment 36. The upstream segment 32 of thestem portion in the present example comprises the protein tag-encodingsequence 14, and the downstream segment 36 comprises the stop codon 16and the ribosome binding site 18. Formation of the stem loop 30 blocksaccess of the ribosome 22 to the ribosome binding site 18 and therebyprevents translation of the response gene 20.

The translation-coupling cassette of the present invention can beincluded in a vector. As used herein, “vector” refers to an entitycomprising the translation-coupling cassette that is capable ofintroducing the translation-coupling cassette into a host fortranscription of the translation-coupling cassette and translation ofany encoded genes. The vector can include nucleic acid sequences thatpermit it to replicate in the cell, such as an origin of replication.The vector can also include a promoter upstream of the target-genecloning site or target gene or other regulatory elements such asenhancers to initiate transcription of the translation-couplingcassette. The vector can further include translational controlsequences, such as a ribosome binding site and/or upstream AT-richsequences, operably connected to the target gene or target gene cloningsite. The vector can also include one or more selectable marker genesindependent of the response gene to isolate hosts harboring the vector.Suitable expression vectors include, but are not limited to viralvectors, such as those based on baculovirus, vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus,and the like; phage vectors, such as bacteriophage vectors; plasmids;phagemids; cosmids; fosmids; bacterial artificial chromosomes; P1-basedartificial chromosomes; yeast plasmids; yeast artificial chromosomes;and any other vectors specific for hosts of interest (such as E. coli,Pseudomonas pisum, or Saccharomyces cerevisiae). Commercially availablevectors for expressing heterologous proteins in bacterial hosts thatprovide elements for inclusion in the vector of the present inventioninclude but are not limited to pZERO, pTrc99A, pUC19, pUC18, pKK223-3,pEX1, pCAL, pET, pSPUTK, pTrxFus, pFastBac, pThioHis, pTrcHis, pTrcHis2,and pLEx.

Some versions of the invention include hosts that comprise thetranslation-coupling cassette, such as hosts that comprise a vectorharboring the translation-coupling cassette. Suitable hosts include anycells or cell-free systems that contain the appropriate molecularmachinery to transcribe the translation-coupling cassette and translatethe genes encoded therein. With respect to translation, the host mustcontain the appropriate molecular machinery for operation with theparticular response-gene translation control element, such as theribosome binding site.

The ribosome binding sites disclosed herein are derived from microbes,and more specifically, prokaryotic microbes. Therefore, the preferredhost is a prokaryotic host. Examples of suitable prokaryotic hostsinclude Gram-positive bacteria such as strains of Bacillus, (e.g., B.brevis or B. subtilis), Pseudomonas, or Streptomyces, or Gram-negativebacteria, such as strains of E. coli. Particularly desirable hosts inthis regard include bacteria that do not produce lipopolysaccharide andare, therefore, endotoxin free.

Although not preferred, the host may also be a eukaryotic host. For theeukaryotic host to employ prokaryotic-derived ribosome binding sites inthe translation-coupling cassette, the host must express the appropriatemolecular machinery. The appropriate molecular machinery may include aprokaryotic ribosome and the initiation factors required for initiationof prokaryotic translation. The appropriate molecular machinery mayfurther include elongation and release factors required for elongationand termination, respectively, of prokaryotic translation. Suitableeukaryotic hosts include such yeast hosts as strains of Saccharomyces,such as S. cerevisiae; Schizosaccharomyces; Kluyveromyces; Pichia, suchas P. pastoris or P. methlanolica; Hansenula, such as H. Polymorpha;Yarrowia; or Candida. Other suitable eukaryotic hosts include suchfilamentous fungal hosts as strains of Aspergillus, e.g., A. oryzae, A.niger, or A. nidulans; Fusarium or Trichoderma. Other suitableeukaryotic hosts include such insect hosts as a Lepidoptora cell line,such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells(“HIGH FIVE”-brand insect cells, Invitrogen, Carlsbad, Calif.) (U.S.Pat. No. 5,077,214). Yet other suitable eukaryotic hosts include suchmammalian hosts as Chinese hamster ovary (CHO) cell lines, e.g., CHO-K1(ATCC CCL-61); green monkey cell lines, e.g., COS-1 (ATCC CRL-1650) andCOS-7 (ATCC CRL-1651); mouse cells, e.g., NS/O; baby hamster kidney(BHK) cell lines, e.g., ATCC CRL-1632 or ATCC CCL-10; and human cells,e.g., HEK 293 (ATCC CRL-1573). Additional suitable cell lines are knownin the art and available from public depositories such as the AmericanType Culture Collection, Rockville, Md.

A suitable host can be produced by introducing the translation-couplingcassette in it. As used herein, “introduce,” used with reference tointroducing the translation-coupling cassette into a host, refers to thedelivery of the translation-coupling cassette to the host in a mannersuch that any genes encoded by the translation-coupling cassette arecapable of being transcribed and translated within the host. Thus,introducing a translation-coupling cassette in a host suitably includesintroducing a vector comprising the translation-coupling cassette in thehost. Introducing the translation-coupling cassette can be performed byboth transformation and transfection. Transformation encompassestechniques by which the translation-coupling cassette is introduced intohosts such as prokaryotic cells or non-animal eukaryotic cells.Transfection encompasses techniques by which the translation-couplingcassette is introduced into hosts such as animal cells. These techniquesinclude but are not limited to introduction of the translation-couplingcassette via conjugation, electroporation, lipofection, infection, andparticle gun acceleration. The introduction of a vector into a bacterialhost may, for instance, be performed by protoplast transformation (Changand Cohen (1979) Molecular General Genetics, 168:111-115), usingcompetent cells (Young and Spizizen (1961) Journal of Bacteriology,81:823-829; Dubnau and Davidoff-Abelson (1971) Journal of MolecularBiology, 56: 209-221), electroporation (Shigekawa and Dower (1988)Biotechniques, 6:742-751), or conjugation (Koehler and Thorne (1987)Journal of Bacteriology, 169:5771-5278). Methods of introducing vectorsinto the eukaryotic hosts are well-known in the art.

Suitable promoters for use in prokaryotic hosts include but are notlimited to: promoters capable of recognizing the T4, T3, Sp6, and T7polymerases; the P_(R) and P_(L) promoters of bacteriophage lambda; thetrp, recA, heat shock, araBAD, propionate, trc, tac, tac-lac, tet,constitutive sigma 70, and lacZ promoters of E. coli, as well as anyartificial promoter selected from engineered libraries (see Alper et al.(2005) PNAS 102(36):12678-83); the alpha-amylase and the sigma-specificpromoters of B. subtilis; the promoters of the bacteriophages ofBacillus; Streptomyces promoters; the int promoter of bacteriophagelambda; the bla promoter of the beta-lactamase gene of pBR322; and theCAT promoter of the chloramphenicol acetyl transferase gene. Prokaryoticpromoters are reviewed by Glick, J. Ind. Microbiol. 1:277 (1987); Watsonet al, Molecular Biology of the Gene, 4th Ed., Benjamin Cummins (1987);and Sambrook et al., In: Molecular Cloning: A Laboratory Manual, 3^(rd)ed., Cold Spring Harbor Laboratory Press (2001).

Suitable promoters for use within a eukaryotic host are typically viralin origin and include, without limitation, the promoter of the mousemetallothionein I gene (Hamer et al. (1982) J. Mol. Appl. Gen. 1:273);the TK promoter of Herpes virus (McKnight (1982) Cell 31:355); the SV40early promoter (Benoist et al. (1981) Nature (London) 290:304); the Roussarcoma virus promoter; the cytomegalovirus promoter (Foecking et al.(1980) Gene 45:101); the yeast gal4 gene promoter (Johnston et al.(1982) PNAS (USA) 79:6971; Silver et al. (1984) PNAS (USA) 81:5951); andthe IgG promoter (Orlandi et al. (1989) PNAS (USA) 86:3833).

Inducible, repressible, and constitutive promoters are all suitable foruse in the present invention. Inducible promoters are those whereinaddition of an effector induces expression. Suitable effectors includeproteins, metabolites, chemicals, or culture conditions capable ofinducing expression. Suitable inducible promoters include but are notlimited to the lac promoter (regulated by IPTG or analogs thereof), thelacUV5 promoter (regulated by IPTG or analogs thereof), the tac promoter(regulated by IPTG or analogs thereof), the trc promoter (regulated byIPTG or analogs thereof), the araBAD promoter (regulated byL-arabinose), the phoA promoter (regulated by phosphate starvation), therecA promoter (regulated by nalidixic acid), the proU promoter(regulated by osmolarity changes), the cst-1 promoter (regulated byglucose starvation), the tetA promoter (regulated by tetracycline), thecadA promoter (regulated by pH), the nar promoter (regulated byanaerobic conditions), the p_(L), promoter (regulated by thermal shift),the cspA promoter (regulated by thermal shift), the T7 promoter(regulated by thermal shift), the T7-lac promoter (regulated by IPTG),the T3-lac promoter (regulated by IPTG), the T5-lac promoter (regulatedby IPTG), the T4 gene 32 promoter (regulated by T4 infection), thenprM-lac promoter (regulated by IPTG), the VHb promoter (regulated byoxygen), the metallothionein promoter (regulated by heavy metals), theMMTV promoter (regulated by steroids such as dexamethasone) and variantsthereof.

Repressible promoters are those wherein addition of an effectorrepresses expression. Examples of repressible promoters include but arenot limited to the trp promoter (regulated by tryptophan);tetracycline-repressible promoters, such as those employed in the“TET-OFF”-brand system (Clontech, Mountain View, Calif.); and variantsthereof.

Constitutive promoters do not require an effector to initiatetranscription. Suitable constitutive promoters are known in the art.

The translation-coupling cassette described herein has many uses. Oneuse comprises a method of assessing whether full-length products oftarget genes are being produced in a host. This method includes thesteps of introducing the translation-coupling cassette into a host anddetermining the presence or level of response-gene expression. The stepof determining the presence or level of response-gene expression isperformed by detecting the presence of the product of the response gene,as described herein.

The translation-coupling cassette can also be used to identify cultureconditions that increase target protein production. This can beperformed by culturing identical hosts in two different cultureconditions and comparing the expression levels of the response geneamong the various conditions. The variables changed in the differentculture conditions may include culture temperatures, particularnutrients, etc.

The translation-coupling cassette can also be used to screen a DNAlibrary for genes that enhance expression of a target gene. Suitable DNAlibraries include, without limitation, genomic, cDNA, and metagenomiclibraries. The method is performed by introducing a host with twovectors—a first comprising the translation-coupling cassette and asecond from a DNA library. Clones from the DNA library that enhancetarget-gene expression can be identified by screening levels ofresponse-gene expression or by selecting hosts that express a selectableresponse gene. The effective clones can be identified by sequencing.Possible factors that may facilitate expression of a target gene mayinclude ribosomal components, chaperones, modifying enzymes,translational enhancers, and/or regulatory proteins. Thetranslation-coupling cassette can also be used to determine if anyrandom sequence of codons will be translated in a chosen host.

The translation-coupling cassette can also be used to select for mutatedversions of a target gene for versions that express or express at higherlevels. This can be performed by generating various versions of thetarget gene by error prone PCR or other known mutagenic techniques,cloning the mutated versions of the target genes intotranslation-coupling cassettes using known techniques, introducing thetranslation-coupling cassettes into hosts, and comparing the expressionlevel of each version of the target gene by determining the respectivelevels of response-gene expression.

Optimization of translation of the target gene may require modificationof any portion or the entirety of the target gene's coding region, 5′UTR, or other elements therein.

In the coding region, the nucleotide sequence of the target gene can bekept the same as that of the wild-type gene found in the genome of thesource organism, or it can be different. Owing to the degeneracy of thegenetic code, changes in the nucleotide sequence of the target gene neednot necessarily lead to changes in the amino acid sequence of the geneproduct.

Codons in the coding region may be optimized to increase expression ofthe target gene in a selected host. One example of optimizing codonsincludes modifying the codon usage of the coding sequence of the targetgene. Although codon usage is not widely believed to impact thetranslational efficiency of an mRNA in higher eukaryotes, a codon biassimilar to that of E. coli does enhance the translational efficiency ofheterologous genes in E. coli. Avoiding infrequently-used codons canalso lead to enhanced stability of an mRNA because sequences thatpromote instability of mRNAs often comprise infrequently-used codons.Codon optimization can be performed for any nucleic acid by“OPTIMUMGENE”-brand gene design system by GenScript (Piscataway, N.J.)or any other commercial or proprietary algorithm.

The coding region of the target gene can also be altered to remove orinsert restriction enzyme sites. Removal of a restriction enzyme site orsites may be required for successful transformation ofrestriction-positive prokaryotic hosts which express the correspondingrestriction enzyme. Removing or inserting restriction enzyme sites canbe performed by site-specific mutagenesis or cassette mutagenesistechniques, which are well known in the art. Regardless, it is generallypreferred when using prokaryotic hosts, to use restriction-minus hosts.

Although nucleotide changes in the coding region do not automaticallylead to changes in the amino acid sequence of the gene product, theamino acid sequence of the gene product can be changed by changing thenucleotide sequence of the target gene by techniques known in the art.In some cases, a particular amino acid sequence is more amenable totranslation in a particular host than other amino acid sequences.

The 5′ untranslated regions (UTRs) of a target gene transcript can alsobe cloned or synthesized, and in either case changed or left intact, bytechniques known in the art. Modifying the 5′ UTR of the target gene mayaffect its translational efficiency. The 5′ untranslated region (UTR)comprises the ribosome binding site and also plays a role in mRNAstability. Use of a ribosome binding site for the target gene dissimilarto the consensus sequence for ribosome binding sites in the host cellcan lead to either reduced rates of translation of the gene product,recognition of cryptic ribosome binding sites elsewhere in the mRNA withsubsequent translation of non-desired truncated or frame-shiftedproteins, or both. To avoid such unwanted effects, the ribosome bindingsite of the 5′ UTR of the target gene can be modified from its native ororiginal sequence to match or nearly match the consensus sequence forthe ribosome binding site in the host cell. Such modification can beaffected by site-specific mutagenesis or cassette mutagenesistechniques, both of which are well known in the art. Also, specificsequences in the 5′ UTR can enhance or diminish the stability of themRNA depending on the host cell, and stabilizing sequences can be addedand destabilizing sequences removed by the site-specific mutagenesis orcassette mutagenesis techniques. 5′ UTR stabilizing and destabilizingsequences in particular host cells are widely known to those of skill inthe art.

While in many versions of the translation-coupling cassette describedherein the target gene encodes a “protein of interest” for downstreampurposes and the response gene serves as an indicator of target geneexpression, these roles can be reversed for particular purposes. In oneversion, a series of translation-coupling cassettes including adetectable (i.e., selectable or screenable) target gene, preferably afluorescent target gene, is constructed. The target gene of eachtranslation-coupling cassette in the series is driven by a differenttranslational control sequence with known relative efficiency. Thetranslation-coupling cassettes further include a response-gene cloningsite for insertion of a gene of interest. With such a system, theresponse gene can be expressed at a particular desired andpre-determined level corresponding to the strength of the translationalcontrol element, which can be verified by a level of emittedfluorescence or other detectable characteristic of the target gene. Insuch versions, a protein tag may be translationally linked to theresponse-gene cloning site for ease in purification of the responsegene.

In yet another version, the target gene may encode a protein of noparticular interest, and the response gene may encode a protein ofinterest. A series of translation-coupling cassettes may be constructed,each having a secondary structure sequence with a differenttranslational control sequence of known relative efficiency controllingthe response gene. As above, a particular translation-coupling cassettecan be chosen for use according to the level of translation of therespone-gene product desired.

Many of the steps described herein for manipulating and analyzingnucleic acids and proteins, including digesting with restrictionendonucleases, amplifying by PCR, hybridizing nucleic acids, ligatingnucleic acids, separating and isolating by gel electrophoresis,transforming cells with heterologous DNA, selecting successfultransformants, purifying with chromatography, performing Western blots,and the like, are well known and widely practiced by those skilled inthe art and are not extensively elaborated upon herein. Unless otherwisenoted, the protocols utilized herein are described extensively inSambrook & Russell (2001), Molecular Cloning: A Laboratory Manual, ThirdEdition; Cold Spring Harbor Laboratory Press: New York, N.Y., ISBN-13:978-0879695774.

The present invention is, in part, directed to DNA translation-couplingcassettes and RNA transcripts thereof. While DNA primarily includesthymine (T) rather than uracil (U), RNA primarily includes U rather thanT. Any disclosure herein of a sequence including T for a DNAtranslation-coupling cassette also constitutes a disclosure of acorresponding sequence with U in place of T for the RNA transcript.Conversely, any disclosure herein of a sequence including U for an RNAtranscript constitutes a disclosure of a corresponding sequence with Tin place of U for the DNA translation-coupling cassette. Unlessexplicitly stated otherwise or indicated otherwise by the context, anygenetic element defined in a DNA cassette, e.g., gene, coding sequence,response-gene translation control element, secondary structure-formingsequence, or protein tag-encoding sequence, applies equally to acorresponding element in the RNA transcript, and vice versa.

“Inserting,” used in reference to inserting a gene into a cloning siterefers to cloning the gene into the cloning site by any method describedherein or known in the art.

“Operably linked” is used herein to refer to joined nucleic acidsequences wherein one sequence performs a regulatory operation onanother sequence. Nucleic acid sequences which are operationally linkedare not necessarily directly contiguous to one another but may beseparated by intervening nucleotides which do not interfere with theoperational relationship of the linked sequences.

“Translationally linked,” used with reference to a first genetic elementand a second genetic element, means that the first genetic element andany intervening sequences between the first genetic element and thesecond genetic element does not include an in-frame stop codon. Thismeans that translation of a first genetic element that istranslationally linked with a second genetic element will comprisetranslation of the first genetic element, any intervening sequencesbetween the first genetic element and the second genetic element, andportions or all of the second element up to the first in-frame stopcodon.

“Gene product” or variations thereof is used herein to refer to thepolypeptide produced by transcription of a specific DNA coding regioninto mRNA followed by translation of the mRNA by a ribosome. Such apolypeptide may also be referred to as a “protein.” If the gene productfunctions as a catalyst in a chemical reaction, the gene product mayalso be referred to as an “enzyme.”

“Upstream” and “downstream” are used herein in the sense commonly usedin the art to refer to directions toward the 5′ end and the 3′ end,respectively, of nucleic acid molecules or toward the N-terminus orC-terminus, respectively, of protein molecules.

The elements and method steps described herein can be used in anycombination whether explicitly described or not. All combinations ofmethod steps as described herein can be performed in any order, unlessotherwise specified or clearly implied to the contrary by the context inwhich the referenced combination is made.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e.,“references”) cited herein are expressly incorporated by reference tothe same extent as if each individual reference were specifically andindividually indicated as being incorporated by reference. In case ofconflict between the present disclosure and the incorporated references,the present disclosure controls.

The tools and methods of the present invention can comprise, consist of,or consist essentially of the essential elements and limitationsdescribed herein, as well as any additional or optional steps,components, or limitations described herein or otherwise useful in theart.

It is understood that the invention is not confined to the particularconstruction and arrangement of parts herein illustrated and described,but embraces such modified forms thereof as come within the scope of theclaims.

EXAMPLES

A series of translation-coupling cassettes with target-gene cloningsites and selection markers as response genes were constructed usingstandard molecular biological techniques. The translation-couplingcassettes and their features are summarized in Table 1, portions ofwhich are shown in FIGS. 2A-E. Plasmid vectors with several of thetranslation-coupling cassettes and one of several different target genescloned in the target-gene cloning site were generated, as summarized inTable 2. The plasmids contained the necessary transcriptional andtranslational control elements for transcription of thetranslation-coupling cassette and translation of the inserted targetgene, respectively. The plasmids were introduced into E. coli cellsusing standard techniques. The transformed cells were cultured in thepresence of various levels of selection agent and assessed for cellgrowth to determine levels of response-gene expression. In addition,target-gene expression levels were assessed by detecting fluorescenceemitted by fluorescent target genes (i.e., red fluorescent protein, RFP)and/or purification of the target-gene product via a fused 6x-His tagusing Ni-NTA affinity chromatography and analysis by SDS-PAGE.

TABLE 1 Translation-Coupling Cassettes^(‡) Target-Gene Response HairpinSEQ ID Name Cloning Site gene Sequence NO TC1 BglII, EcoRI; Cm^(R) FIG.2A 1 TC2 BglII, EcoRI; Cm^(R) FIG. 2B 2 ligation-independent cloningcompetent TC3 SpeI, MfeI; Cm^(R) FIG. 2C 3 TC4 SpeI, MfeI; Kan^(R) FIG.2D 4 TC5 SpeI Kan^(R) FIG. 2E 5 ^(‡)Cm^(R), chloramphenicol resistancemarker; Kan^(R), kanamycin resistance marker

TABLE 2 Plasmids with Various Translation-Coupling Cassettes ComprisingTarget- Gene Inserts and Observed Phenotypes of Strains HarboringThem^(‡) Translation- Target- Coupling Gene Plasmid Cassette InsertPhenotype Observed pTC1-RFP TC1 RFP Growth in all Cm concentrations;strong fluorescence; strong RFP protein band (FIGS. 3A-C) pTC1-RFP* TC1RFP* Weak growth in low Cm; no detectable fluorescence (FIGS. 4A-B)pTC2-RFP TC2 RFP Growth and fluorescence trends inversely with Cmconcentration (FIGS. 5A-B) pTC2-RFP* TC2 RFP* No growth; no fluorescence(FIGS. 6A-B) pTC4-RFP TC4 RFP Growth and fluorescence trends inverselywith Km concentration (FIGS. 7A-B) pTC4-RFP* TC4 RFP* No growth; nofluorescence (FIGS. 8A-B) pTC4-D1-D8 TC4 D1-D8 of No growth (FIG. 10)Gene X pTC4-D1-D2 TC4 D1-D2 of Growth; purified protein Gene X (FIGS.11A-B) pTC4-D3-D5 TC4 D3-D5 of Growth; purified protein Gene X (FIGS.12A-B) pTC4-D6-D8 TC4 D6-D8 of No growth; no purified protein Gene X(FIGS. 13A-B) ^(‡)RFP, full-length red fluorescent protein; RFP*, RFPwith premature stop codon; Cm, chloramphenicol; Km, kanamycin

As a proof of concept for the expression-coupling system describedherein, a first set of experiments was carried out with the TC1translation-coupling cassette using either full length red fluorescentprotein (RFP) gene or an RFP gene with a premature stop codon (RFP*)inserted as the target gene. Vectors harboring each cassette weredesignated as pTC1-RFP and pTC1-RFP*, respectively (see Table 2). E.coli cells transformed with each plasmid vector were grown in thepresence of 34, 61, 89, 116, 144, 171, or 199 μg/ml chloramphenicol.

Cells transformed with pTC1-RFP, i.e., the full RFP coding sequence,showed strong growth (FIG. 3A) and strong RFP fluorescence (FIG. 3B) atall chloramphenicol concentrations. This indicated that both the RFPtarget protein and the chloramphenicol resistance marker were fullytranslated. Analysis by SDS-PAGE showed presence of the RFP proteinproduct at the correct molecular weight (FIG. 3C), verifying fulltranslation of the RFP protein. By contrast, cells transformed withpTC1-RFP*, i.e., the RFP coding sequence with the premature stop codon,showed essentially no growth (FIG. 4A) and no fluorescence (FIG. 4B).

These experiments show that expression of the response gene in thecurrent expression system is coupled to full expression of the targetgene and can be used to select for transformed cells that translate thetarget gene in its entirety.

A second set of experiments was carried out with the TC2translation-coupling cassette with the same two RFP inserts as describedabove. The TC2 translation-coupling cassette had a differenthairpin-forming sequence than that of TC1 (compare FIGS. 2A and 2B).Plasmid pTC2-RFP included the TC2 translation-coupling cassette with thefull-length RFP coding sequence, and plasmid pTC2-RFP* included the TC2cassette with the truncated RFP coding sequence. In cells harboringpTC2-RFP, both growth (FIG. 5A) and fluorescence (FIG. 5B) trendedinversely with increasing chloramphenicol concentration. By contrast,cells harboring pTC2-RFP* showed no growth (FIG. 6A) or detectablefluorescence (FIG. 6B). These results indicated that translation of theresponse gene (Cm^(R)) was coupled to the translation of the target gene(RFP).

As with the first set of experiments, these data show that expression ofthe response gene in the current expression system is coupled to fulltranslation of the target gene and can be used to select for transformedcells that translate the target gene in its entirety.

A third set of proof-of-concept experiments was performed with the TC4translation-coupling cassette. The TC4 translation-coupling cassetteincluded the same secondary structure-forming sequence as TC2 butcontained a kanamycin-resistance marker in place of a chloramphenicolmarker (see FIGS. 2B and 2D and Table 1). The same two RFP genes used inthe previous set of experiments were cloned into the target-gene cloningsite to generate pTC4-RFP and pTC4-RFP* (Table 2). E. coli cellstransformed with either pTC4-RFP and pTC4-RFP* were cultured in thepresence of 0, 12.5, 25, 50, 100, and 200 μg/ml kanamycin. Both growth(FIG. 7A) and fluorescence (FIG. 7B) of cells harboring pTC4-RFP trendedinversely with increasing kanamycin concentration. By contrast, cellsharboring pTC4-RFP* displayed no growth in the presence of kanamycin(FIG. 8A) and no detectable fluorescence (FIG. 8B).

These data again show that expression of the response gene in thecurrent expression system is coupled to full translation of the targetgene and can be used to select for transformed cells that translate thetarget gene in its entirety. Further, these experiments show that thesystem can be used with any of several different response genes.

In a fourth set of experiments, the translation-coupling system was usedto assess expression of a previously uncharacterized gene (“Gene X”) inE. coli. Gene X encodes a protein comprising eight predicted domains(FIG. 9) and, at about 9 kb, is much larger than usual E. coli genes. Totest expression of Gene X in E. coli, the full-length gene was clonedinto the target-gene cloning site of the TC4 translation-couplingcassette, and the latter was inserted into a plasmid to generatepTC4-D1-D8 (Table 2). The pTC4-D1-D8 plasmid was used to transform E.coli and the transformed cells were cultured in the presence of 50, 100,150, 200, 250, and 300 μg/ml kanamycin. The cells failed to grow at allconcentrations of kanamycin (FIG. 10), indicating that the full-lengthgene did not successfully express.

In light of the failed growth of the full-length gene, smaller (1-3 kb)sections of Gene X based on the domain architecture of the protein wereused as target genes and tested for expression using the same protocolas for the full-length gene. A gene fragment encoding roughly theN-terminal third of the protein (Domains 1-2) was used to generatepTC4-D1-D2 (Table 2). Cells harboring this plasmid successfullytranslated the D1-D2 polypeptide, as evidenced by growth in kanamycin(FIG. 11A) and SDS-PAGE analysis of the His-tagged, Ni-NTA-purifiedprotein fragment (FIG. 11B). Similarly, a gene fragment encoding roughlythe central third of the full-length protein (Domains 3-5) was used togenerate pTC4-D3-D5. This fragment also successfully translated (FIGS.12A and 12B). A gene fragment encoding roughly the C-terminal third ofthe protein (Domains 6-8) was used to generate pTC4-D6-D8 (Table 2).Cells harboring this plasmid did not successfully translate thefragment, as evidenced by a lack of growth in kanamycin (FIG. 13A; thecurve at 300 μg/ml kanamycin appears to be a contaminant) and lack of adetectable band with SDS-PAGE (FIG. 13B). Future experiments will useerror-prone PCR to generate D6-D8 fragments for use as target genes inthe present system to identify mutant genes that can be efficientlyexpressed.

This series of experiments shows that the translation-coupling cassettesdisclosed herein can be used to assess expression levels of a previouslyunknown, uncharacterized, and unexpressed target gene by selecting orscreening for activity of the response gene. This system is amenable foruse in a high-throughput system and real-time detection of target-geneexpression.

Future experiments will study the relationship between expression levelof the target gene and resistance generated by expression of theantibiotic resistance marker. This will be performed by usingtranslation start sequences of varying efficiency (predicted by Salis etal. Nature Biotechnology (2009) 27:946-950) for RFP as a target gene,and monitoring both RFP fluorescence and RFP abundance by Western blot.We predict that levels of antibiotic resistance will correlate withlevels of fluorescence and RFP abundance.

1. A translation-coupling cassette comprising: a target gene or atarget-gene cloning site configured for forming a target gene; aresponse gene or a response-gene cloning site configured for forming aresponse gene; a response-gene translation control element that, in atranscript of the translation-coupling cassette, controls translation ofthe response gene or a response gene formed with the response-genecloning site; and a secondary structure-forming sequence that, in thetranscript of the translation-coupling cassette, reversibly forms asecondary structure that masks the response-gene translation controlelement, wherein at least part of the secondary structure-formingsequence is translationally linked with the target-gene cloning site orthe target gene.
 2. The translation-coupling cassette of claim 1 whereinthe target-gene cloning site or the response-gene cloning site includesa multiple cloning site.
 3. The translation-coupling cassette of claim 1wherein the target-gene cloning site or the response-gene cloning siteincludes a ligation-independent cloning site.
 4. Thetranslation-coupling cassette of claim 1 further comprising a proteintag-encoding sequence, wherein the protein tag-encoding sequence istranslationally linked with the target-gene cloning site ortranslationally linked in-frame with the target gene.
 5. Thetranslation-coupling cassette of claim 1 wherein the response gene is ascreenable gene.
 6. The translation-coupling cassette of claim 1 whereinthe response gene is a selectable gene.
 7. The translation-couplingcassette of claim 1 wherein the response-gene translation controlelement is a ribosome binding site.
 8. The translation-coupling cassetteof claim 7 wherein the ribosome binding site comprises a Shine-Dalgarnosequence or derivative thereof.
 9. The translation-coupling cassette ofclaim 7 wherein the ribosome binding site comprises an AT-rich sequence.10. The translation-coupling cassette of claim 1 further comprising alinker disposed between the response-gene translation control elementand the response gene or the response-gene cloning site.
 11. Thetranslation-coupling cassette of claim 1 further including a stop codonupstream, within, or downstream of the secondary structure-formingsequence, the stop codon being translationally linked with thetarget-gene cloning site or translationally linked in-frame with thetarget gene.
 12. The translation-coupling cassette of claim 1 whereinthe secondary structure formed by the secondary structure-formingsequence in the transcript includes a stem loop.
 13. Thetranslation-coupling cassette of claim 1 wherein the secondarystructure-forming sequence at least partially includes the response-genetranslation control element.
 14. The translation-coupling cassette ofclaim 13 wherein the secondary structure-forming sequence furtherincludes a stop codon, the stop codon being translationally linked withthe target-gene cloning site or translationally linked in-frame with thetarget gene.
 15. The translation-coupling cassette of claim 14 whereinthe secondary structure-forming sequence at least partially includes aprotein tag-encoding sequence, wherein the protein tag-encoding sequenceis translationally linked with the target-gene cloning site ortranslationally linked in-frame with the target gene.
 16. Thetranslation-coupling cassette of claim 1 wherein the secondarystructure-forming sequence is selected from the group consisting ofbases 18-46 of SEQ ID NO:1, bases 26-60 of SEQ ID NO:2, bases 26-60 ofSEQ ID NO:3, bases 26-60 of SEQ ID NO:4, and bases 5-51 of SEQ ID NO:5.17. The translation-coupling cassette of claim 1 wherein thetranslation-coupling cassette is included within a vector capable ofbeing introduced in a host.
 18. The translation-coupling cassette ofclaim 1 further including a host comprising the translation-couplingcassette.
 19. The translation-coupling cassette claim 18 wherein thehost is a prokaryote.
 20. An RNA molecule formed by transcription of atranslation-coupling cassette as recited in claim
 1. 21. A method ofassessing expression including: introducing a translation-couplingcassette into a host, wherein the translation-coupling cassettecomprises: a target gene; a response gene; a response-gene translationcontrol element that, in a transcript of the translation-couplingcassette, controls translation of the response gene; and a secondarystructure-forming sequence that, in the transcript of thetranslation-coupling cassette, reversibly forms a secondary structurethat masks the response-gene translation control element, wherein atleast part of the secondary structure-forming sequence istranslationally linked with the target gene; and determining a level ofresponse-gene expression.
 22. The method of claim 21 further includinggenerating a mutated version of the target gene; cloning the mutatedversion of the target gene into a second translation-coupling cassette;introducing the second translation-coupling cassette into a second hostof same type as the host; determining a level of response-geneexpression in the second host; and comparing the level of response-geneexpression in the host with the level of response-gene expression in thesecond host.
 23. The method of claim 21 further including, prior to thedetermining step, culturing the host in a plurality of cultureconditions, wherein the determining the level of response-geneexpression includes determining a level of response-gene expression foreach of the plurality of culture conditions, and further includingcomparing the levels of response-gene expression for each of theplurality of culture conditions.
 24. The method of claim 21 wherein theintroducing step further includes introducing and expressing a clonefrom a DNA library.
 25. (canceled)
 26. The translation-coupling cassetteof claim 1 wherein the at least the part of the secondarystructure-forming sequence is translationally linked with thetarget-gene cloning site or the target gene such that the response geneis translationally coupled to the target gene or a target gene formedwith the response-gene cloning site.